This proposal has two separate overall objectives. The first concerns a study of the special properties of a chemically modified crosslinked hemoglobin (HbXL) with a greatly enhanced capability for oxygen delivery even at low temperature. The molecular reasons for these remarkable properties will be explored by means of measurements of both equilibria and kinetics of ligand binding, and the stability of the heme-globin linkage as reflected by heme transfer to hemopexin. A special effort will be devoted to a detailed comparison of the hydrogen exchange kinetics of HbXL and HbA since a great deal of knowledge is already available in the case of normal hemoglobin. The possible biomedical usefulness of HbXL will be tested on two different models. In one case the recovery from hemorrhagic shock in rats who have lost 2/3 of their own blood volume will be assessed. The other application will involve the reduction by HbXL infusion of the neurologic deficit which results from experimental ischemia in the spinal cord of rabbits. The second objective concerns the interaction of hemoglobin with allosteric cofactors, especially polyglutamates of folates and antifolates. The binding of these polyanions will be studied under physiological conditions, i.e. in the presence of a great excess of hemoglobin and as a function of such modalities as oxygen pressure, 2,3-DPG concentration and mutations in the protein. The role of hemoglobin for the storage of folates or methotrexate in mature red cells and its possible role in the regulation of folate enzymes in erythrocyte precursors will also be explored.